Aureobasidium pullulans metabolism of prostaglandins of the A-type

$\text{Aureobasidium pullulans}$, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that $\text{A. pullulans}$ can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). $\text{A. pullulans}$ metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. $\text{A. pullulans}$ displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type.

Aureobasidium pullulans, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA2 (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9 alpha or 9 beta-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9 alpha, 15 alpha-dihydroxy-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9 beta-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9, 15-oxo-2, 3, 4, 5-tetranorprostanoic acid and 9, 15-oxo-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. The water soluble metabolites have not been characterized further. The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that A. pullulans can perform the following transformation: beta-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both delta 10,11 and delta 13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (delta 10, 11 leads to delta 11, 12). A. pullulans metabolizes 15-epimeric PGA2 equally readily with the production of similar products. PGA1 affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. A. pullulans displays some enantioselectivity, PGA2 and 15-epi-PGA2 are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Preparation and quantitation of urinary metabolites of prostaglandin F2α by radioimmunoassay

Radioimmunoassay systems are described which have been developed to quantitate two principle urinary metabolites of PGF_2α; 9α,11α-dihydroxy-15-oxo-2,3,4,5-tetranorprostanoic acid (I) and 9α-11α-dihydroxy-15-oxo-2,3,4,5-tetranorprosta-1,20-dioic acid (II). Preparation of the required metabolites was achieved by total synthesis (I) or by bioconversion (isolation from urine of animals treated with 15-keto-PGF_2α^*, II). These metabolites were used to prepare conjugates for immunization. Labeled metabolites, suitable as binding markers, were prepared by metabolism of ³H-PGF_2α (I) or (II). Specificity of the resulting antibodies was compared to an antibody to PGF_2α and to 13,14-dihydro-15-keto PGF_2α. Antisera of II had little or no affinity for 20-carbon precursors (PGF_2α or 13,14-dihydro-15-keto PGF_2α), but had nearly equal affinity for metabolite I. Antisera of I, however, had little or no affinity for antigen of II. Therefore, analysis of samples by both assay systems enables quantitation of these excretion products of PGF_2α. Other assay parameters (binding, affinity, recovery, precision and the repeatability of the assays) were similar to those previously described for other RIA systems, and were considered satisfactory for quanitation of compounds in biological fluids.Quantitation of 24 hour urinary excretion of di-acid metabolite in humans was in close agreement with previously published values determined by physical-chemical means. Greater quantity of di-acid metabolite was excreted by human males (42.0 μg/24 hr) than by females sampled either during the follicular (20.0) or luteal phase (21.2) of the menstrual cycle. The total quantity of C-16 metabolites (as approximated by system II) excreted/kg body weight by the rhesus monkey was similar to that excreted by the human. However, the ratio of di-acid to mono-acid was much nearer unity in the monkey than the human.     

Synthesis of β-oxidation products as potential leukotriene metabolites and their detection in bile of anesthetized rat

Two novel β-oxidation products of peptido leukotrienes, 16-carboxy-17, 18, 19, 20-tetranor-14, 15-dihydro-N-acetyl LTE₄ and 18-carboxy-19, 20-dinor-N-acetyl LTE₄, were prepared by total synthesis and used to identify previously unknown polar rat biliary metabolites. When [³H] LTC₄ and synthetic N-acetyl-LTE₄ were administered intravenously to anesthetized inbred male rats, extraction of the bile and subsequent reverse-phase HPLC fractionation allowed the isolation of two novel metabolites of N-acetyl-LTE₄. Comparison of U.V. spectra and coelution experiments revealed that these metabolites correspond to the above-mentioned synthetic β-oxidation products. This was further confirmed by the coelution of the corresponding methyl esters. Oxidative ozonolysis of the metabolically produced 16-carboxy-17, 18, 19, 20-tetranor-14, 15-dihydro-N-acetyl LTE₄ (major metabolite) confirmed the absence of the 14, 15-unsaturation. The presence of these metabolites indicates that peptide leukotrienes undergo N-acetylation followed by ω and subsequent β-oxidation in the anesthetized rat.     

High performance liquid chromatography and UV detection for the separation and quantitation of prostaglandins

A fast and reliable method for the separation and quantitation of arachidonic acid metabolites PGF_1α, PGF_2α, PGD₂, PGE₁, PGE₂, PGB₂, PGA₂, 6-keto PGE₁, 6-keto PGF_1α, T×B₂ and 15-keto PGE₂ by high-performance liquid chromatography has been developed. Utilizing a single reverse-phase column and a UV spectrophotometer, sensitivity as little as 30 nanograms of each of these prostaglandins can be separated and subsequently detected. Although this study was performed using standards, it is highly promising for future application to biological fluids.     

In vivo formation of β-oxidized metabolites of leukotriene E4 in the rat

Intraperitoneal administration of [³H]-leukotriene E₄ in the rat resulted in the appearance of radiolabel in urine and feces. Separation of polar urinary metabolites and chromatographic comparison of synthetic metabolites indicated the in vivo formation of ω-oxidized metabolites of LTE₄ with sequential β-oxidation. Futhermore, the metabolite identified as 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl-LTE₄ substantiates the biochemical patheway of β-oxidation in vivo involving the 2,4-dienoyl CoA reductase as an integral step. These results substantiate β-oxidation of sulfidopeptide leukotrienes in vivo and these metabolites account for some of the major urinary metabolites of this class of lipid mediator.     

Prostaglandin A1 induces differentiation in friend erythroleukemia cells

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B₂ and 6-keto PGF₁α, were completely inactive, while PGE₁ inhibited slightly and PGF₂α stimulated the replication of FLC. PGA₁ was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA₁ alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA₁-stimulated differentiation was completely suppressed by the addition of 10⁻⁶M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA₁ (but no DMSO) prevented the return to the undifferentiated state.     

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF_1α was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF_1α was recovered in the urine. The metabolic pattern very closely resembles that of PGI₂ in rats. Metabolites were found which represented 15-dehydrogenation, β-oxidation, ω and ω-1-hydroxylation and oxidation.Previous work showed that 6-keto PGF_1α is very poorly oxidized by 15-PGDH. We administered 15-[H³]-PGI₂ and 15-[H³]-6-keto PGF_1α to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI₂ and 6-keto PGF_1α were both oxidized to the 15-keto product, although the rate of oxidation of PGI₂ was greater than that of 6-keto PGF_1α. We concluded that the administered PGI₂ was oxidized by 15-PGDH before hydrolysis to 6-keto PGF_1α. A portion of the dose is probably hydrolyzed before 15-dehydrogenation.     

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a ³²P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE₁, PGE₂ and to a less extent by PGF_2α and PGA₂. In the case of PGE₂, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE₁, PGE₂ and PGF_2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE₂. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.     

Luteolytic prostaglandins synthesis and biological activity

Analogues of PGF_2α with enhanced luteolytic activity were synthesized using the Corey synthesis. The luteolytic activity of the new prostaglandins was tested in the hamster.In addition the smooth muscle activity of the new compounds was compared with that of PGA₂ on the longitudinal strip of rat stomach fundus. Structure-activity relationships in the new series of 17,18,19,20-tetranor-16-thienyl-oxy-PGF_2α are discussed.     

Metabolism and excretion of exogenous [H]-LTC4 in primates

Four novel ω- and β-oxidation (from the ω end) products of peptide leukotrienes, 20-hydroxy and 20-carboxy-LTE₄, 18-carboxy-19,20-dinor-LTE₄ and 16-carboxy-17, 18, 19, 20-tetranor-14, 15-dihydro-LTE₄ were prepared by total synthesis and used as standards for identification of biliary and urinary metabolites in the cynomolgus monkey. After intravenous administration 14, 15,-[³H] leukotriene C₄ (10 μCi kg⁻¹ was partially metabolized in and rapidly cleared from the vascular circulation. This resulted, within 24 hours, in significant urinary excretion. (14.8 ± 2.1%, n = 4), consisting largely of material more polar than LET₄ (61% of urinary excretion) as shown by reverse phase HPLC. The polar fraction demonstrated two predominant metabolites which coeluted in several HPLC solvent systems with synthetic 16-carboxytetranordihydro-LTE₄ (major component) and 18-carboxydinor-LTE₄ (minor component). Characterization of the major polar metabolites as 16-carboxytetranordihydro-LTE₄ was substantiated by conversion to its N-acetylated derivative. The absence of the 14, 15 double bond was confirmed by product analysis of oxidative ozonolysis. In a single animal, the bile duct was cannulated, with significant biliary excretion of radioactivity demonstrated over 4 hours (58.6% recovery). The predominant polar biliary metabolites were also identified as the 18-carboxydinor and 16-carboxytetranordihydro derivatives of LTE₄ mentioned above. These data suggest that β-oxidation products generated from the ω-carboxyl end of the 20-carboxyl-LTE₄ are important products of [³H] LTC₄ metabolism in the monkey. Quantitation of these urinary metabolites may be an important index of leukotriene production.     

Metabolism of prostaglndin A. II. Isolation, characterization, and synthesis of PGA1 renal.

Since the renal cortex has recently been shown to be a major site of prostaglandin A₁ (PGA₁) metabolism, studies were undertaken to isolate and characterize the major metabolites. Homogenates of rabbit cortex (500g) were incubated with ³H-PGA₁ (50mg) in the presence of NAD⁺ (50mg). Acidic lipid extracts were subjected to linear gradient silicic acid chromatography. Six radioactive peaks were recovered, of which peak 4 was unconverted PGA₁. The major metabolites (1,3) were further subjected to reversed phase partition chromatography and TLC with and without silver nitrate. Three PGA₁ analogs were then synthesized via oxidation of the secondary alcohol group at C-15 by manganese dioxide (15-keto-PGA₁). The second compound was synthesized by hydrogenation of 15-keto-PGA₁ (15-keto 13, 14-dihydro PGA₁). The third compound (13, 14-dihydro PGA₁) was obtained by direct catalytic hydrogenation of PGA₁. Purification of these substances were achieved by a combination of silicic acid and thin layer chromatography. It was found that metabolite 1 cochromatographed on TLC (AgNO3) with synthesized 15-keto 13, 14-dihydro PGA₁. Both compounds were 100 times less potent than PGA₁ in lowering rat blood pressure. Metabolite 3 cochromatographed on TLC (AgNO3) with synthesized 13, 14-dihydro PGA₁. Both were as potent as PGA₁ in lowering rat blood pressure. Metabolites 1 and 3 absorbed UV at 221 nm but not at 280 nm following alkali treatment. These studies suggest that rabbit renal cortex metabolizes PGA₁ to what appears to be biologically active 13, 14-dihydro PGA₁ and biologically inactive 15-keto 13, 14-dihydro PGA₁. It remains possible that the hypotensive effect of PGA₁ is the result of its conversion to its biologically active 13, 14-dihydro derivative.     

Effects of various prostanoids on th in vitro metabolism of bovine articular chondrocytes

The dose-dependent effects of 9 prostanoids (PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂, PGI₂, 6 keto- PGF_1α) on metabolism of cultured bovine articular chrondrocytes were investigated. Most prostanoids dose-dependently inhibited ³⁵SO₄⁼ and ³H-glycine incorporation. At 25 μg/ml, the inhibitory sequence was A₂D₂>E₂ = E₁ = A₁>6 keto-F_1α>F₁>F₂, but sensivity (lowest dose eliciting inhibition) followed the sequence E₂ > 6 keto-F_1α = F₁ > A₂ = D₂>E₁>A₁. At 25 μg/ml PGA₂ also inhibited incorporation of ³H-cytidine and ^#H-thymidine, but had no significant effect on ³H-glucose or ¹⁴C-xylose incorporation. The inhibitory effect of PGA₂ was apparent after 30 minutes exposure for ³⁵SO₄= and after 60 minutesd for ³H-cytidine, and was still present up to 72 hours following incubation in fresh non-PG-containing medium. PGI₂ had no significant effect of ³⁵SO₄⁼ incorporation but at concentrations below 10 μg/ml enhanced uptake of ³H-glycine.The PG-induced inhibitory effect was apparently not due to cell damage as indicated by measurement of ³H-glucose metabolism and lactate production.     

Effect of prostaglandins on H-thymidine uptake into human epidermal cells

Prostaglandins A₂, B₁, E₁, E₂, F_1α and F_2α were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37°C, and the effects on ³H-thymidine uptake into DNA was measured. At 70 μg/ml all prostaglandins tested except PGF_2α inhibited the uptake of ³H-thymidine greater than 50%. However, at 35 μg/ml, PGA₂ and PGB₁ were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 μg/ml only PGA₂ caused substantial inhibition, 68%. In order to determine if the PGA₂ action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA₂. None of the above receptor antagonists were able to reduce the PGA₂-induced inhibition of ³H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of ³H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

Antihypertensive activity of 16,16-dimethyl-oxa-alkyl-prostaglandins of the PGA2, PGE2 and trans-Δ 2-11-deoxy-PGE1 series: Structure-activity relationships

Utilizing Corey's synthesis, a variety of prostaglandins (PGs) with a modified ω-side chain were prepared. The 16,16-dimethyl-oxa-alkyl analogues of PGA₂ had potent antihypertensive activity. HR 466 (16,16-dimethyl-18-oxa-PGA₂), the best compound out of this series was active for 5–6 hours after oral administration of 0,1 mg/kg to conscious renal hypertensive dogs. The corresponding analogues of PGE₂ were also potent antihypertensive compounds, but were much more spasmogenic. Structural variations within the trans-Δ2-11-deoxy-PGE₁-series, in both side chains, gave HR 601 (trans-Δ2-15α-acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester) which was orally active in the hypertensive dog with similar activity to HR 466.     

Intracellular glutathione level modulates the induction of apoptosis by Δ-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an α, β-unsaturated carbonyl of 9-deoxy-Δ^9,12-13,14-dihydro PGD₂ (Δ¹²-PGJ₂), on the cytotoxicity of Δ¹²-PGJ₂. Δ¹²-PGJ₂ caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Δ¹²-PGJ₂-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by Δ¹²-PGJ₂. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of Δ¹²-PGJ₂ is due to thiol-reactivity and intracellular GSH modulates the Δ¹²-PGJ₂-induced apoptosis by regulating the accessibility of Δ¹²-PGJ₂ to target proteins containing thiol groups.     

Prostaglandin specific binding in hamster myometrial low speed supernatant

A charcoal adsorption method was developed to measure specific prostaglandin binding in low speed supernates of hamster myometrial homogenates. This method was used to characterize and quantitate PGE₁-specific binding. The equilibrium binding constants and the concentration of specific PGE₁ binding sites were determined during the hamster estrous cycle. The apparent association constant for 12 different preparations was 1.16 ± 0.08 × 10⁹M⁻¹. The concentration of PGE₁ specific binding sites was significantly higher on Days 2 and 3 of the estrous cycle than it was on Days 1 or 4. The competition for PGE₁ binding sites by PGE₂, PGF_2α, PGA₁ and various PGE₁ metabolites and derivatives was measured in hamster myometrial homogenates. Relative affinities of the natural prostaglandins for the PGE₁ binding sites, calculated by parallel line assay, were: PGE₂>PGE₁>PGA₁>PGF_2α. For PGE₁ metabolites the relative affinities were: PGE₁>13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁>15-keto-PGE₁. For the analogs and derivatives the compounds tested ranked as: 16,16-dimethyl-PGE₁≥PGE₁>PGE₁ methyl ester>17-phenyl-18,19,20-trinor-PGE₁>15(S)15-methyl-PGE₁ methyl ester. Arachidonic acid, bis-homo-γ-linolenic acid and 7-oxa-13 prostynoic acid had relative affinities ≥0.1 compared to PGE₁=100. Indomethacin had a relative affinity of 0.4 compared to PGE₁.     

The chemical structure of prostaglandin X (prostacyclin)

The chemical structure of prostaglandin X, the anti-aggregatory substance derived from prostaglandin endoperoxides, is 9-deoxy-6, 9α-epoxy-Δ⁵-PGF₁α. The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems is 6-keto-PGF₁α. Prostaglandin X is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9α-epoxy-Δ⁵-PGF₁α.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

Effect of PGI2, PGE2 and 6-keto PGF1α on canine gastric blood flow and acid secretion

Prostaglandins (PG)I₂, PGE₂ and 6-keto PGF₁α were infused directly into the gastric arterial supply at 10⁻⁹, 10⁻⁸ and 10⁻⁷ g/kg/min during an intra-gastric artery pentagastrin infusion in anesthetized dogs. 6-keto PGF₁α was also infused at 10⁻⁶ g/kg/min. Gastric arterial blood flow was measured continuously with a non-cannulating electromagnetic flow probe and gastric acid collected directly from the stomach. PGI₂ and PGE₂ produced similar dose-dependent increases in blood flow with an increase of more than four-fold at the highest dose. Both PGs inhibited acid output over this dose range with PGE₂ having 10 times the potency of PGI₂. 6-keto PGF₁α was at least 1000 times less active than PGI₂ or PGE₂ at increasing blood flow and failed to inhibit acid output even at 10⁻⁶ g/kg/min.